Nicotinic acetylcholine receptors (nAChRs), the ligand gated ion channels at which nicotine acts, play an important role in nicotine abuse and understanding its neurobiology may be central to developing more rational approaches for nicotine abuse treatments. The in vitro characterization of the receptor-ligand interaction is an important step in understanding the action of a ligand in vivo (i.e., for interpretation of the results from the functional studies of receptors). To analyze the ligand-receptor interaction at physiological conditions, a competition receptor assay at 37 degrees C was developed. This assay used [I-125]-5-iodo-A-85380 (5IA), which was developed and characterized by staff of the Neuroimaging Research Branch as a high affinity, selective radioligand for alpha4 beta2 nAChRs, to determine the affinities of 28 nicotinic agonists and antagonists (9 established, 8 previously and 11 recently developed in our chemistry lab) at 23 degrees C and 37 degrees C. The Kd values for these ligands ranged from 0.01 to 25,000 nM. Increasing incubation temperature from 23 to 37 degrees C resulted in variable increases in the observed Kd values (from 1.0 to 3.3-fold) and variable effects on the kinetics of receptor-ligand interaction. For example, the association and dissociation of the receptor-ligand complex for 5IA were approximately 10 times faster at 37 degrees C than that at 23 degrees C, but the rate of [H-3]-cytisine binding kinetics increased only 3.5 fold. This variability in effects of temperature on receptor binding properties suggests that characterization of ligand-receptor interaction at physiological temperature may better predict the in vivo properties of the ligands and may be important in the interpretation of results from the functional studies of alpha4 beta2 nAChRs. Previous studies from Abbott Laboratories, where Me-p-PVC (5-(2-(4-pyridinyl)vinyl)-6-chloro-3-(1-methyl-2-(S)-pyrrolidinylmethoxy)pyridine) was developed, demonstrated that this compound had high affinity for alpha4beta2 nicotinic acetylcholine receptors (nAChRs) and attenuated nicotine-induced efflux of 86Rb from IMR-32 cells, contained alpha3beta4 nAChRs. In vitro binding studies with Me-p-PVC showed that its affinity for alpha4beta2 nAChRs exceeded that for the alpha3beta4 receptor subtype by a factor of 3,000 (Kd ca. 0.025 and 90 nM, respectively). Substantial anecdotal evidence suggests that nicotine may function as a gateway drug to illicit drug use. As such we utilized an animal behavorial model of drug rewards conditioned place preference (CPP) to test the hypothesis that nicotine produces behavioral cross-sensitization to different classes of abused drugs, specifically opiates, stimulants and marihuana. These experiments in rats demonstrated that nicotine pretreatment enhances the rewarding effects of amphetamine for at least 3 to 5 days following the cessation of nicotine, with this effect dissipating within 19 days. The underlying mechanism involves alpha4beta2 nAChRs as the competitive alpha4beta2 antagonist dihydro beta erythroidine effectively blocked the development of nicotine-induced cross sensitization. Interestingly, the alpha7 nicotinic antagonist methyllycaconitine also antagonized cross-sensitization at doses that do not block nicotine self-administration in rats. This study and published report have clearly demonstrated that nicotine produces cross-sensitization to the rewarding effects of both opiates and psychostimulants measured with CPP. The development of cross-sensitization to the rewarding effects of these drugs involves the interaction of nicotine with both alpha4beta2 and alpha7 nAChRs, which differs from nicotine self-administration, which is primarily mediated by alpha4beta2 receptors.